Journal: Nature Communications

Article Title: A Cas9 with PAM recognition for adenine dinucleotides

doi: 10.1038/s41467-020-16117-8

Figure Lengend Snippet: a Sequence alignment of SpyCas9, its QQR variant, and SmacCas9. The step in the underlining red line marks the joining of SpyCas9 and SmacCas9 to construct a SpyMac hybrid. The sequence logo (Weblogo online tool) immediately below the alignment depicts the conservation at 11 positions around the PAM-contacting arginines of SpyCas9. b The domain organization of SpyCas9 juxtaposed over a color-coded structure of RNA-guided, target-bound SpyCas9 (PDB ID 5F9R). The two DNA strands are black with the exception of a magenta segment corresponding to the PAM. A blue–green–red color map is used for labeling the Cas9 PI domain and guide spacer sequence to highlight structures that confer sequence specificity and the prevalence of intra-domain contacts within the PI . c A sequence logo generated online (WebLogo) that was input with putative PAM sequences found in Streptococcus phage and associated with close SmacCas9 homologs.

Article Snippet: The gBlock (IDT) encoding the PAM-interaction domain of S. macacae was inserted into a bacterial protein expression/purification vector containing wild-type S. pyogenes Cas9 fused to the His6-MBP-tobacco etch virus (TEV) protease cleavage site at the N-terminus (pMJ915 was a gift from Jennifer Doudna, Addgene plasmid #69090).

Techniques: Sequencing, Variant Assay, Construct, Labeling, Generated

Journal: Nature Communications

Article Title: A Cas9 with PAM recognition for adenine dinucleotides

doi: 10.1038/s41467-020-16117-8

Figure Lengend Snippet: a CRISPResso2 indel analysis following NGS of amplified genomic regions to assess on-target editing of iSpyMac in comparison to SpyMac and SpyCas9, on the indicated 5 \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$^{\prime}$$\end{document} ′ -NAA-3 \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$^{\prime}$$\end{document} ′ and 5 \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$^{\prime}$$\end{document} ′ -NGG-3 \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$^{\prime}$$\end{document} ′ PAM sequences. Samples were performed in two independent transfection replicates ( n = 2). b Efficiency heatmap of mismatch tolerance assay on genomic targets. Quantified indel frequencies, as assessed by the TIDE algorithm , are exhibited for each labeled single or double mismatch in the sgRNA sequence for the indicated Cas9 variant and indicated PAM sequence. Samples were performed in two independent transfection replicates ( n = 2). c CRISPResso2 genomic base editing analysis following NGS of genomic amplicons to assess conversion of cytosines to thymines by iSpyMac-BE3. Samples were performed in two independent transfection replicates ( n = 2). All source data are provided as a Source Data file.

Article Snippet: The gBlock (IDT) encoding the PAM-interaction domain of S. macacae was inserted into a bacterial protein expression/purification vector containing wild-type S. pyogenes Cas9 fused to the His6-MBP-tobacco etch virus (TEV) protease cleavage site at the N-terminus (pMJ915 was a gift from Jennifer Doudna, Addgene plasmid #69090).

Techniques: Amplification, Comparison, Transfection, Labeling, Sequencing, Variant Assay